Granulocyte colony stimulating factor (known also as GCSF, G-CSF, CSF3, or colony stimulating factor-3) is a monomeric secreted protein belonging to the IL-6 protein superfamily. G-CSF acts in hematopoiesis as a cytokine that controls the production, differentiation, and function of granulocytes, a related white cell population of the blood.
The human G-CSF gene contains 5 exons and 4 introns and is located on chromosome 17. Two different polypeptides of molecular weight 19,600 are synthesized from the same gene by differential mRNA splicing (Nagata et al., 1986; Souza et al., 1986; Metcalf, 1985). The two polypeptides differ by the presence (long form) or absence (short form) of 3 amino acids. Expression studies indicate that both the long and short form have G-CSF activity biological activity.
The two forms of G-CSF (designated CSF3_HUMAN) are described in Swissprot databank: one is a polypeptide of 204 amino acids (the “isoform short”, P09919-2) and the other is a polypeptide of 207 amino acids (the “isoform long”, P09919-1).
G-CSF is a secreted protein; comprising a signal peptide represented by amino acid residues 1 to 30. The resulting matured “short” and “long” isoforms have respectively 174 and 177 amino acids. The mouse orthologue of CSF3_HUMAN is CSF3_MOUSE (Swissprot databank). It is found on mouse chromosome 11. The genomic organization of the mouse gene is similar to that of the human with 5 exons and 4 introns. No alternatively spliced forms have been reported for the mouse gene.
G-CSF has two intra-molecular disulfide linkages between Cys-36 and Cys-42, and between Cys-64 and Cys-74, with a free cysteine residue at position 17. It also contains an O-glycosylation site at Thr-133 (all numbering is done with respect to the mature (lacking the signal peptide) short form of the polypeptide). The protein can be glycosylated with O-glycan consisting of Gal-GalNAc disaccharide, which can be modified with up to two sialic acid residues (done in recombinantly expressed G-CSF from CHO cells).
The secondary structure of human G-CSF is dominated by alpha-helical regions with four stretches of helices. The four stretches of helices have been identified between residues 11 and 41 (helix A), 71 and 95 (helix B), 102 and 125 (helix C), and 145 and 170 (helix D), which form a left-handed four-helix bundle with helices A and B aligned parallel to one another (up-up) and antiparallel to helices C and D (down-down). An additional short fifth helix (E) is part of the AB loop connecting helices A and B.
hG-CSF and related mutants or variants cDNAs and proteins have been disclosed in EP0243153. Splicing variants of G-CSF have been reported in R. Sorg, J. Enczmann, U. Sorg, K. Heermeier, E. M. Schneider, and P. Wernet. Rapid and sensitive mRNA phenotyping for interleukins (IL-1 to IL-6) and colony-stimulating factors (G-CSF, M-CSF, and GM-CSF) by reverse transcription and subsequent polymerase chain reaction. Exp Hematol JID—0402313 19 (9):882-887, 1991; Cusi M. Grazia and D. Ferrero. Harlequin granulocyte-colony stimulating factor interleukin 6 molecules with bifunctional and antagonistic activities. Immunotechnology JID—9511979 3 (1):61-69, 1997; and WO03027288A1. Analogs of human G-CSF have been generated by mutagenesis or by fusion with heterologous sequences immunogenicity half-life (WO 04/020576; WO 02/020767; WO 02/020766; WO 02/066514; WO 02/077034; WO 03/076567; WO 02/069232; WO 01/073081; WO 99/58662; WO 96/39422; WO 95/21254; WO 95/13393; WO 95/33057; WO 92/06116; WO 90/12874; EP272703; EP459630; EP243153; U.S. Pat. No. 4,904,584; U.S. Pat. No. 4,810,643; AU 76380/91; AU 10948/92). Also, non-natural variants of human G-CSF have been generated to improve their activity by mutagenizing specific residues and linking non-peptide moieties such as PEG molecules (WO 03/031464; WO 03/006501; EP401384; EP473268; EP335423; U.S. Pat. No. 5,824,778; U.S. Pat. No. 5,824,784). Furthermore, antibodies against human G-CSF have been described (EP0331186).
G-CSF activates a receptor of the hematopoietin receptor superfamily, the G-CSF receptor (G-CSF-R), which subsequently triggers multiple signalling mechanisms. Four isoforms of G-CSF-R are described in Swissprot (Accession no Q99062), but seven human G-CSF-R isoforms seem to exist, all generated by alternative splicing of a single receptor gene transcript. Some of the isoforms of G-CSF-R seem to have tissue specificity.
The G-CSF-R forms a tetrameric complex with G-CSF, said complex comprising two ligand and two receptor molecules. The NH2 region (residues 20-46) and the COOH terminal region (including helix D) of G-CSF are involved in binding to the receptor. Different studies agreed with the presence of one receptor-binding site involving various residues on the helices A and C. Some reports suggested the existence of a second binding site located on the helix E (1997, PMID 9194183; 2003, PMID 12946100). The most important residue for receptor binding is Glu 19 (in helix A). Other quoted residues are Lys 40, Glu 46 (helix E) and Phe 144 (helix D). In addition to these residues, Val 48, Leu 49 (helix E), Leu 15 (helix A), Asp 112 and Leu 124 (helix C) seem to be important for biological activity.
G-CSF is available as a drug under different names (Neupogen, Granulokine, or Granocyte) for the treatment of neutropenia, a disorder characterized by an extremely low number of neutrophils in blood, as an adjunct to chemotherapy, bone marrow transplantation, and collection of CSF-mobilised peripherical blood progenitors cells.
The efficacy of G-CSF in decreasing the risk of infections in Felty's syndrome and systemic lupus erythematosus (SLE) has also been well documented (Chronic neutropenia with autoimmune diseases is associated mainly with rheumatoid arthritis as Felty's syndrome or large granular leukaemia, and with SLE).
The use of G-CSF for the treatment of myelogenous leukemia has been described in EP0231819.
G-CSF also provides therapeutic effect in fighting infections, by causing selective activation of distinct early response genes through different JAK-STAT signalling molecules.
G-CSF has also been described as having anti-apoptotic activity through protein synthesis-dependent mechanisms involving the Janus kinase-STAT pathway.
G-CSF also finds therapeutic utility in clinical transplantation, since this protein induces the mobilization of hematopoietic progenitor stem cells (HPCs) (HPCs ensure the continuous renewal of mature blood cells). In this respect, Petit et al. (2002) investigated the mobilization of hematopoietic progenitor stem cells (HPCs) induced by GCSF. ELISA and immunohistologic analysis showed a significant reduction in SDF1 (CXCL12; 600835) in human and mouse bone marrow plasma and immature osteoblasts, but not peripheral blood, within 24 hours of GCSF treatment. Manipulation of SDF1-CXCR4 interactions may be an improved way to control the navigation of progenitor cells between the bone marrow and blood.
Levesque et al. (2003) demonstrated that the mobilization of HPCs by granulocyte colony-stimulating factor or cyclophosphamide was due to the disruption of the CXCR4/CXCL12 chemotactic pathway. The mobilization of HPCs coincided in vivo with the cleavage of the N terminus of the chemokine receptor CXCR4 found on HPCs. This resulted in the loss of chemotactic response of the HPCs to the CXCR4 ligand, CXCL12. The concentration of CXCL12 was also decreased in vivo in the bone marrow of mobilized mice, and this decrease coincided with the accumulation of serine proteases capable of direct cleavage and inactivation of CXCL12. As both CXCL12 and CXCR4 are essential for the homing and retention of HPCs in the bone marrow, the proteolytic degradation of CXCL12 and CXCR4 may represent a critical step in the mobilization of HPCs into the peripheral blood by GCSF or cyclophosphamide.
G-CSF has also been associated with the expression of proliferation vascularization in meningioma.
Moreover, a significantly higher level of G-CSF has been found in follicular fluid than in serum. Combined with the fact that G-CSF and its receptor are expressed in follicular fluid by granulosa cells, it has been suggested that G-CSF plays an important role in ovarian function. Other results demonstrate that G-CSF is produced in the human follicle shortly before the ovulatory phase and may play a role in the mechanism of ovulation.
G-CSF also seems to have immunomodulatory function in host defence. G-CSF administration has a number of effects that might simultaneously enhance host defence while reducing the risk of developing uncontrolled systemic inflammation. This may also be efficacious in prolonging graft survival and reducing graft versus host disease. Significant associations of endogenous G-CSF levels with anti-inflammatory mediators early in the development of severe lung injury suggest an endogenous anti-inflammatory role of G-CSF in vivo.
Recombinant G-CSF induces production of anti-inflammatory factors and is protective against endotoxin- and sepsis-induced organ injury. It has been suggested that rG-CSF may be beneficial as an adjunct therapy for treatment of serious bacterial and opportunistic fungal infections in normeutropenic patients, including those with alterations in neutrophil function. Endogenous G-CSF increases neutrophil function in patients with severe sepsis and septic shock, necessary for resolution of bacterial infections in these patients (2002, PMID 12027409).
It has been reported that G-CSF improves cardiac function and reduces mortality after acute myocardial infarction (Myocardial infarction is accompanied with an inflammatory reaction which induces cardiac dysfunction and remodelling). Takano et al (2003, PMID 12769752) suggested that G-CSF may regenerate cardiac myocytes and blood vessels through mobilization of bone marrow stem cells.
Recombinant G-CSF induces production of anti-inflammatory factors and is protective against endotoxin- and sepsis-induced organ injury (Root et al., 1999). Hartung (1999, PMID 10596675) indicated that, in addition to its anti-infectious role, G-CSF has an immunomodulatory function and also augments antibiotic efficacy. Important uses of G-CSF in oncology are prevention of febrile neutropenia after chemotherapy, treatment of febrile neutropenic episodes and support following bone marrow transplantation, and collection of CSF-mobilised peripherical blood progenitors cells (Dale, 2002, PMID 12479591).
Considering the biological activities of G-CSF, it would be highly valuable to obtain biologically active G-CSF variants, particularly agonists of human G-CSF.